Sub-project #1 Analysis of biological samples using immunoaffinity capillary electrophoresis (ICE) Capillary electrophoresis combined with lased induced fluorescent (LIF) detection is an established method for analyzing biological samples. The small sample volumes, short analysis times and low reagent use are advantageous over more traditional methods such as HPLC. Derivatization of the capillary with antibodies allows for specific capture and detection of single or multiple analytes from a complex biological sample. ICE increases the limits of detection for CE without the need for pre-processing samples. Additionally this technique can be used on both a traditional CE machine as well as a micro-chip CE. ICE has been used to process samples from several collaborators on the NIH campus. Studies include the following: -Analysis of human plasma samples for cytokines indicated in kidney diseases -Tissue analysis from mice and rats from disease model studies looking at Alzheimer's Disease -Analysis of tissue samples from rats given cytokine inhibition drugs Sub-project #2 Multiplex ELISA analysis of cell secretions Multiplex ELISA improves upon the traditional ELISA format. Rather than detecting a single antigen in a single sample, multiplex technology allows for the detection of up to 16 antigens from a single sample. This reduces the overall time and cost associated with ELISA and requires far less sample volume to produce more information. The multiplex format is customizable to allow researchers to investigate a unique array of proteins and peptides. Studies using multiplex ELISA include: -Analysis of mouse plasma samples from mice involved in social defeat models -Analysis of human plasma samples from subjects involved in sleep stimulation trials -Analysis of cell secretion products from Hela cells stimulated by UV light and cold. Sub- project #3 MALDI mass spectrometry of proteins MALDI mass spectrometry is a technique used to pinpoint the mass of analytes ranging from a few hundred mass units to tens of KDa units. The technique is a complement to our isolation techniques, allowing us to pinpoint the mass of unknown analytes isolated from complex samples. This technique allows researchers to positively identify isolated unknowns and determine elemental composition. Studies using MALDI include: -Analysis of IgA isolated from rhesus macaques for the presence of J-chain component -Analysis of fluorescent proteins EGFP and M-Ruby-2 for precise molecular weights of cleavage products Sub-project #4 Biacore Biacore 3000 detection system uses Surface Plasmon Resonance (SPR) to monitor the refractive index (RI) change as molecules interact at the sensor chip surface. SPR is the excitation of surface plasmons by light. One of the interactants (ligand) is immobilized on the sensor chip surface, while the other (analyte) is injected in continued flow over the surface. If the interaction takes place, the surface concentration of the analyte is increased that results in the RI change. SPR technology enables label-free real-time detection and monitoring of biomolecular events and provides qualitative and quantitative information on specificity of binding between two molecules, affinity, and kinetics of the interaction. Experimental design is highly flexible and the technology can be applied to protein interaction with other proteins, nucleic acids, lipids, small molecules. Studies using Biacore include: -Analysis of the interaction of the Kd of Her- protein with DNA aptamers Sub-Project #5 SELEX by capillary electrophoresis SELEX is technique by which DNA aptamer libraries are screened for unique DNA sequences that show an affinity for a certain protein. The aptamers are then collected and amplified by PCR and the selection is repeated until an enriched pool of aptamers showing affinity for the protein is obtained. This procedure is time and material consumming. Capillary electrophoresis has been used to increase the sensitivity of this process and decrease the amount of time the procdure takes. Studies using SELEX_CE include - Screening DNA libraries for aptamers that show an affinity for Her-2 protein - Screening DNA libraries for aptamers that show an affinitiy for MIG protein